Red nocardia cell wall skeleton preparation process and its therapeutic use on treating cervical erosion

ABSTRACT

This invention relates to a biological preparation, especially  Nocardia Rubra  cell wall skeleton preparation. After cultivating, collecting thallus, cell fragmentation, purifying enzyme, removing the grease, the final preparation containing  Nocardia Rubra  cell wall skeleton is obtained by dispensing, lyophilizing and filling. The invented preparation has marked effect to adaptable diseases, healing rate is 88%, total validity rate is 100%. This external preparation shows harmless, safe, non-toxicity and easy to be used with little side effect and lower cost.

TECHNICAL FIELD

The present invention is related to a kind of microbial preparationproduced by bioengineering technology, which is a preparation ofNocardia Rubra cell wall skeleton. The main component of the preparationis Nocardia Rubra cell wall skeleton that contains Arabic semi-glactan,muramic acid and mucopeptide etc. Moreover, the invention is alsorelated to the process and detective method of the preparation, as wellas its effect on the treatment of cervical erosion.

BACKGROUND OF THE INVENTION

Cervical erosion is the most common chronic disease in gynecology, andabout 30 percent distaff of pregnant age are suffering from it. Thesymptom may be increased leucorrhea, lumbosacral pain and bearing down.If the local is inflammatory, pyogenic leucorrhoea will appear, andtherapy must be carried now. As long as severe cervical erosion is notdealt with in time, it will easily lead to canceration.

At present, the physical therapy generally used includes electrotherapy,freezing, laser and microwave etc. But the methods mentioned above willbring the patient variety of side effect, such as injury of normaltissue, excessive vaginal secretion accompanied by bad smell.Simultaneously, the effect is not satisfactory and also constrained byseason. It is not suitable to apply them for therapy in summer.

Nowadays, the medicine we frequently use in dealing with the patientincludes embrocation, embolism, effervesce and the like. But medicineshave many shortcomings, e.g. un-effectiveness, high relapse probability,a lot of side effects and even tend to break the microbial ecologicalbalance of internal environment in vagina.

In a word, ideal therapy has not been found out to deal with cervicalerosion now. Most patients have no choice but to endure the pain causedby these therapies.

SUMMARY OF INVENTION

In order to overcome the shortcoming of the prior arts, the object ofthe present invention is to provide a kind of Nocardia Rubra cell wallskeleton preparation and its preparation method.

Another object of this invention is to apply the preparation containingNocardia Rubra cell wall skeleton in the treatment of cervical erosion.

The invented preparation has distinct effects on indication, less sidereaction, no obvious local irritating symptom and no injury to normaltissue. At the same time, it can lead to deceased secretion, improvedproperty and warranted safety. It is for convenient use with low cost.

To carry out the objects, the invention provides a kind of preparationto deal with cervical erosion, characterized by containing NocardiaRubra cell wall skeleton.

The preparation can also contain the pharmaceutically acceptablecarriers. The Nocardia Rubra had been deposited in China GeneralMicrobiological Culture Collection Center in Feb. 5, 2002 withdeposition No.: CGMCC. No. 0712

The pharmaceutically acceptable carriers comprise saccharide(monosaccharide, disaccharide, polysaccharide), glycoride (e.g.Dextran), fat (fatty acid included), protein( e.g. albumin, gelatin),amino acid (e.g. sodium Glutamate, glycine, cysteine) and the like, andlipid (e.g. polysorbate), alcohol (glycerine, propylene glycol,mannitol), dimethyl sulfoxide organic solvent, hydroxyl-carboxy acid(e.g. sodium citrate), polyanion (e.g. Polyphosphate), antisepsis,antioxidation etc, but dextran is preferable.

Based on the total weight of the preparation, about 0.005-99 weightpercent is Nocardia Rubra cell wall skeleton.

The invention also provides with a method of the preparation,comprising: Obtaining product of cell wall skeleton after culture,collection, cell shattering, enzyme purification, delipidation ofNocardia Rubra with deposit No. CGMCC. No. 0712;

Mixing the production of Nocardia Rubra cell wall skeleton and thepharmaceutically acceptable carriers to obtain a preparation.

During the course of preparation, dextran is preferably pharmaceuticallyacceptable carrier. Based on the total weight of the preparation,Nocardia Rubra cell wall skeleton is in amount of about 0.005-99 weightpercent.

The present invention also provides a treatment method of cervicalerosion, characterized by applying pharmaceutically effective amount ofNocardia Rubra cell wall skeleton preparation directly on a sufferingsite of a subject.

Microbe Required in the Invention

The bacteria strain used to produce the preparation of in the inventionbelongs to Nocardia Rubra which possess the ability to produce NocardiaRubra cell wall skeleton preparation. Nocardia Rubra Nr-8206 has theabove properties and is suitable to produce the preparation. Aftercultured 5 days at 33C on glycerine agar media, the bacteria will beobtained, and it had been deposited in China General MicrobiologicalCulture Collection Center in Feb. 5, 2002, deposit No. CGMCC. No. 0712.

Bacterial Property of the Bacteria in the Invention

1. Culture Property

The bacterium, Nocardia Rubra is inoculated on glycerine agar media ofpH 7.2˜7.5, and cultured for 48 hours at 33° C., the bacterial colonywill be eminent and appear orange, and dry with granular appearance, anda bit luster can be seen. Inoculation ring is vulnerable to touch, andmycelial body formation cannot be seen under the laboratory condition.

2. Morphological Property of Bacterium

The bacterium has positive Gram-staining and negative anti-acidstaining. The bacterium is branch shaped with transverse membrane,forming thin mycelia body. The whole mycelia split to irregular columnshaped short thick cell, after cultured for five days, it will appearshort rod-like and spherical.

3. Biochemical Reaction

The bacterium shows fermentation of mannitol, sorbate; and nofermentation of lactose, maltose, sucrose, synanthrin, rhamnose,arabinose, muscarinose, gossypose, semi-lactose, amylum gelatin as wellas positive of nitrate reduction.

Production would be made if above criteria could be met with culture andextract method.

By the way of microbial production, the bacteria in the invention withNocadia Rubra genus can be used. After secondary culture, amplification,ultrasound waves shattering, cell wall skeleton is extracted, followedby enzyme purification, delipidation. Adding appropriate amount ofexcipient, then the culture will be made by freezing to dry. Both solidand liquid culture can be applied in the present invention.

There is no special requirement for nutritional source in culture media,it may contain carbon, nitrogen source and other nutritional sourcesthat generally used in microbial culture. Carbonic sources can be suchas amylum, amylin, mannital, sucrose, lactose, sorbate, maltose, andetc. Nitrogen sources can be meat extract, peptone, ammonium, nitrateand other organic or inorganic nitrogenic compound. Some inorganic saltto other source of nutrition, e.g. phosphate can be included alsooptionally.

There's no strict requirement for culture condition, e.g. time andtemperature whenever culture condition that conductive to the growth ofbacteria and high output is used. For instance, pH level shouldfluctuate around neutralization, culture temperature being about 22˜37°C. Of course, the components, concentration of hydrogen ion, culturetemperature should be adjusted according to different bacteria andexternal condition etc to obtain the best outcome.

By the way of microbial culture, the bacteria in the invention areinoculated on glycerine agar media to culture. After further culture ofqualified bacteria, collection, cell shattering, enzyme purification anddelipidation, the active component cell wall skeleton can be obtained.Then pharmaceutically acceptable carrier known by the person skilled inthe art, e.g. acceptable excipient, preferably such as dextran may beadded, followed by freezing it to be become a commercial product aftergrouting.

The dosage of the product in this invention may be, but not limited, 0.5ml per bottle.

Component and proportion may be as following: Nocardia Rubra cell wallskeleton 5-1000 μg Dextran 0.01-100 mg

By detection of the bacteria strain, culture liquid, the index of thequality of finished product can be seen in the following tables.Examination of bacterial strain: Content of test Criteria of test Resultof test Characteristics The strain is swelling up, The strain isswelling up, of cultivation yellow, with the surface yellow, with thesurface dry, wrinkle, sand granules- dry, wrinkle, sand granules- likeand luster. like and luster. The inoculate ring is fragile The inoculatering is fragile and will not form the aerial and will not form theaerial mycelium in the lab. mycelium in the lab. MorphologicGram-staining is positive, Gram-staining is positive, character of andacid-fast staining is and acid-fast staining is thallus negative.negative. Thallus is branching and Thallus is branching and havingdiaphragm, which is having diaphragm, which is forming the finemycelium. forming the fine mycelium. The whole mycelium is The wholemycelium is divided into irregular divided into irregular short columnshape cell, short column shape cell, and will turn into short and willturn into short stem shape and spherical stem shape and spherical shapeafter 5 days culture. shape after 5 days culture. Biochemical It canferment manicol, It can ferment manicol, reactions sorbitol; cannotferment sorbitol; cannot ferment lactose, maltose, sucrose, lactose,maltose, sucrose, inulin, rhamnose, inulin, rhamnose, arabinose,adonitoxolo- arabinose, adonitoxolo- genin, mannose, gossupose, genin,mannose, gossupose, galactose, starch, animal galactose, starch, animalglue; nitrate reduction glue; nitrate reduction examination is positive.examination is positive.

Examination of stock solution: Content of test Criteria of test Resultof test Determination of total amount of 0.9%˜1.4% 0.9%˜1.4% solidDetermination of sugar content ≧2.5 mg/ml ≧2.5 mg/ml Determination ofmuramic acid ≧200 μg/ml ≧200 μg/ml Determination of protein remnant≦30%  ≦30%  Determination of lipid remnant ≦5% ≦5 Determination of RNAand DNA ≦5% respective ≦5% respective remnant Determination ofTritonX-100 ≦5% ≦5% remnant Aseptic test Negative Negative

Examination of final product: Content of test Criteria of test Result oftest Physical Character The production is white White loose bodies loosebodies or power Moisture content ≦6% 2.74% Solubility Dissolve in 1minute Certified after the addition of 0.9% sodium chloride solution.Sugar differential test Solution is blue-green Solution is blue-greencolor color Content of muramic ≧1.0 μg/bottle 1.64 μg/bottle acidAbnormal toxicity Mice should be alive Certified experiment of miceduring the observation with no abnormal reaction, and the weight of miceshould increase by the end of the observation. Abnormal toxicity Cavyshould be alive Certified experiment of during the observation cavy withno abnormal reaction, and the weight of cavy should increase by the endof the observation Efficacy experiment Phagocytosis percentage:Phagocytosis ≧10% percentage: 43.6% Phagocytosis index: Phagocytosisindex: ≧0.15 0.63 Aseptic test Negative Negative Conclusion of Certifiedexamination

Compared with other available techniques, this invention is thebiological preparation using microbial cell wall skeleton as itseffective component, which is a kind of immunopotentiator. Thus it willhave the anti-tumor function of organism, prevent the infection fromsome virus and bacteria, and have phagocytosis ability of macrophage.This is identified by a series of experiments and clinical trials. Thetreatment effect is satisfactory with the healing rate reached to 88%,general effective rate reached to 100%. And it has no injury to normaltissue, non-toxicity, safety and little side effects. It is also a kindof external preparation for cervical erosion with high efficiency, goodsafety, convenient and low cost.

Nocardia Rubra used in this invention was already deposited in ChinaGeneral Microbiological Culture Collection Center on February 5, 2002with the Deposit serial number: CGMCC No. 0712. And the test showed thatthe preserved strains are alive with no inactivation.

1. General Pharmacological Test for the Invented Product:

1). The blood pressure, respiratory, heart rate and ECG of anestheticcats had no obvious change after the cats were intravenous injected bythe preparation whose dosage was 20, 40 and 80 times of clinical dose.

2). The coordination exercise and memory function of mice had no obviouschange after the mice were intravenous injected by 0.5 ml preparationwhose dosage was 1000 times of clinical dose.

So the preparation of this invention has shown that no obvious effect onthe psychoneurous system, cardiovascular system and respiratory systemof animal are found substantially.

2. Safety test: (Aseptic, Toxicity)

1). Result of aseptic test is negative; identifying the aseptic test iscertified.

2). Acute toxicity test of mice:

The mice in trial group were subcutaneous or abdominal injected by thepreparation whose dosage is five times of human. And the mice incontrast group were injected by the aseptic saline instead. After 7-8days observation, the condition of mice was normal, their weight wasincreasing, and the necropsy had no abnormal findings. So the toxicitytest was also certified.

3. Long Term Toxicity Test:

After three months of administration to this preparation by vagina (30times of clinical dosage), the trial dogs remained normal conditions. Notoxicity on dogs was found and the ECG, hemanalysis index of dogsremained normal, so did two weeks later, which showed that no delayedtoxicity reaction existed.

4. Stability Test of the Preparation:

Under the same condition of technology and in normal temperature,compared with the quality on output date, the content of alanine andmuramic acid in the preparation had no obvious difference after placingfor 1, 2, 3, 8, 14 and 21 months. This shows the good stability of thepreparation. And from the efficacy test, the phagocytosis percentage andindex also has no obvious change. After lyophilized, the preparation canbe preserved for two years in normal temperature.

5. Immunity Test:

The ability of surface immunophagocytosis of this preparation is verypotential, with the phagocytosis percentage ≧10%, phagocytosis index≧0.15. In fact the phagocytosis percentage and index of dextran are evenlower than saline, this shows the potential immunophagocytosis abilityof this preparation which dextran and saline have not.

EXAMPLE

The glycerine-agar culture medium is formed by 2.0-6.0 g of beefextract, 4.0-10.0 g of peptone, 2.0-6.0 g of sodium chloride, 10.0-20.0g of agar, 4.0-10.0 ml of glycerine, 0.1-0.5 g of Na₂HPO₄.12H₂O, and 500ml-1000 ml of distilled water. After sterilization under pH 7.0-8.0,Nocardia Rubra Nr-8206, CGMCC No. 0712 is inoculated into this culturemedium for two-eight days in 28-36° C., to have a culture, then afterwashing off the lawn by aseptic distilled water, the culture will becentrifuged at 1000-5000 rpm for 5-40 minutes, following by collectingbacterial and washing 1-7 times, weighing the wet weight and preservingit in −70° C.-20° C. After the purification test, the culture having noother mixed bacteria can be used. 1-5 portion wet bacteria is oscillatedwith 1-5 portion aseptic distilled water, and shattering them byultrasound disintegrator to have a diluted culture. To takegram-staining and examination by microscope every 5-30 minutes, it willbe certified at least 5 visual fields each has less than 15 tangiblebacteria. Then such culture is got rid of the residual by 1000-2000 rpmcentrifugation for 5-40min, and the supernatant is preserved in −70°C.-20° C. The phosphate PBS buffer solution in PH 7.0-8.0 is wellprepared and preserved after sterilization. And then the cell wallskeleton is extracted. 100-500 ml supernatant is centrifuged at10000-20000 rpm for 5-40 minutes, followed by excluding supernatant andmixing the sediment with PBS buffer solution which contains DNA and RNAenzyme of 100-500 μg/ml in 15-30° C. for 0.5-3 hours to get a mixture.Then the mixture is further centrifuged at 10000-20000 rpm for 5-40minutes, and followed by washing the sediment 1-5 times by PBS buffersolution. The sediment is then tested by RNA and DNA enzyme digestiverate, diluted to 100-500 ml by 0.5-5% polyethylene glycol nonylphenylether for 12-48 hours in 15-30° C. It is centrifuged at 10000-20000 rpmcentrifugation for 5-40 minutes and disposing a supernatant again to getthe another sediment. Such sediment is washed by PBS buffer solution for1-5 times. The sediment is diluted by the solution which containspronase (50-200 μg/ml) and trypsin (1.0-5.0 mg/ml) to 100-500 ml for8-24 hours in 18-30° C., and then continuously centrifuged at10000-20000 rpm for 5-40 minutes to get a sediment again. The sedimentis also washed by PBS buffer solution for 1-5 times. Accordingly 0.5 mlsample to test the protein remnant is kept well.

In the enzyme purification, the enzyme known by those skilled in the artcan be used to cut the protein or peptide of the Nocardia Rubra cellwall into desired pieces for the preparation.

Get Rid of Lipid by Organic Solvent:

The sediment finally obtained as mentioned above is diluted by acetoneto 100-500 ml for 12-48 hours in 18-30° C., and then centrifuged at10000-20000 rpm for 5-40 minutes to get sediment, followed by washing byPBS buffer solution for 1-5 times. Then the sediment is diluted by thesolution which contains diethylether and ethyl alcohol 1:0.5-1:5 to100-500 ml for 12-18 hours in 18-30° C., and further centrifuged at10000-20000 rpm for 5-40 minutes to get sediment again. Such sediment isalso washed by PBS buffer solution for 1-5 times. The lipid remainingrate is tested.

After above steps, the sediment will be the cell wall skeleton. Thesediment is weighed the wet weight and diluted with 10-100 mg/ml ofaseptic distilled water. After sterilization, it is preserved in −70°C.-10° C.

The canned lyophilized procedure: (To dispense 5000 ml semi-manufacturedgoods for example)

The semi-product is composed of 50-1000 mg Nocardia Rubra cell skeleton,0.1-1000 g dextran, aseptic injective water 5000 ml. Then thesemi-product is mixed by magnetic blender. 0.5 ml to every bottle iscanned and then lyophilized well. The finished product is tested toensure that every bottle contains 5-1000 μg Nocardia Rubra cell wallskeleton, 0.01-100 mg dextran. The product can be preserved 2 years innormal temperature.

Clinical Trial:

The clinical trial of this preparation has been taken in the Woman andChildren hospital in Shenyang, China and the results were given asfollows:

A. Research Method: Opened and Multiple-Central Randomized ContrastStudy.

A-A. Requirements of Tested Objects:

-   -   1. Age was between 23 and 45, married and non-pregnant woman.    -   2. Cervical erosion, excluding the cancerization ones.    -   3. Menstruation was regular and menstrual cycle was not shorter        than 25 days.    -   4. able to persist to finish the therapeutic course.    -   5. Not take other therapies during tested phase.

A-B. Exclusion Requirements of Tested Objects:

-   -   1. The result of vaginal cellular smear-inspection was over        II_(B).    -   2. The cervix was cancerization.    -   3. With various vaginal inflammations or acute, sub-acute pelvic        inflammatory diseases.    -   4. During postpartum 3-months.    -   5. With allergic history to many drugs.

A-C. The Number of Tested Objects:

-   -   75 cases in all, and were distributed according to 2:1        randomized table: 50 cases in trial group, and 25 cases in        contrast group.

A-D. Drugs Used and Usage:

1. Trial Group:

The drug was an preparation of Nocardia Rubra cell wall skeleton. Itcould be made into embrocation. The norm was that every bottle had 60 μgfrozen-dry-powder of Nocardia Rubra cell wall skeleton (short forNr-CWS), which was the invention of applicant's company. The batchnumber was 950321.

Usage: After eliminating the excretion on the cervical surface, the 60μg frozen-dry-powder of the embrocation related above was dissolved in2.0 ml physical saline, the aseptic bandage and hygienic cotton ballswhich have been soaked by the drug was put on the cervical erosion area.The patient took them out 24-hours later. The application was repeatedas above twice every week, and 6 times in all.

2. Contrast Group:

The therapeutic drug was a kind of ALBOTHYL concentrate. There was 360mg ALBOTHYL in 1 g of drug, which was a product of BYK Gulden D-78467Konstanz, Germany. The batch number was: J940475.

Usage: Firstly, 1:5 dilution-fluid was used to wash the cervix, and thegauze-clot was used to eliminate the cervical mucus and intra-vaginalexcretion. Then stick cottons were soaked with concentration-fluid tothe erosion areas closely. The patient would take them out 3 minuteslater. At this time, the erosion areas had all turned white. Applicationwas repeated as above 2 times a week, and 6 times in all.

The tested objects of trial group and CONTRAST group all had the therapy2-3 days after the menstruation was over.

A-E. The Diagnostic Criterion

1. Grading:

-   -   Mild degree: Erosion area is smaller than ⅓ of all cervical        area.    -   Moderate degree: Erosion area is account for ⅓ to ⅔ of all        cervical area.    -   Severe degree: Erosion area is over ⅔ of all cervical area.

2. Classification: According to the Depth Degree of Erosion:

-   -   Simple type: The surface of erosion is smooth.    -   Granular type: The surface of erosion is protruding in papilla        form, not flat.

A-F. Observing Items:

-   -   1. Before using drugs:        -   2) Inquired the history and did gynecological examination            conventionally.        -   3) Examined the clean-degree of vaginal secretion, which was            classified into I, II, III degree. And examined Trichomonas            and mold.        -   4) Carried on cellular-smear examination to exclude            cancerization.        -   5) Measured the cervical erosion area by eye. Grading and            classify the erosion according to diagnostic criterion.        -   6) Did blood and urine routine tests. Examined hepatic            function (GPT or ALT) and renal function (BUN, Cr).        -   6) Arranged special people to proceed the therapy and            observation in order to unify the criterion.    -   2. During the phase of using drugs:        -   1) Suggested patients not do sexual intercourse during            therapeutic phase.        -   2) Inquired systemic or local adverse reactions of the drug.        -   3 ) Observed local adverse reactions of vulva and vagina and            change of leucorrhoea (character and amount) attentively            every time before using drugs.    -   3. Post-Therapy:

The patients were reexamined after the next menstruation and continuedto be asked symptoms and to observe local changes of cervical erosion(including area and depth) and the changes of amount and character ofvaginal excretion. The therapeutic effect was evaluated. After the lasttime of using drug, the vaginal cellular examination and laboratoryexamination were repeated (blood and urine routine tests, hepatic andrenal function tests).

A-G The Criterion of the Therapeutic-Effect-Judgment:

Cure: Erosion areas disappear, cervix is smooth, and symptoms disappear.

Effective: The erosion areas of superior and inferior labium contractsby 2 mm, and become more superficial. Symptoms are lightened.

No effect: Erosion areas are larger and deepen. Symptoms are severe.

A-H. Data Management and Statistical Methods:

United programs, united tables and united data managements as well asstatistical method: X² test were done.

Results:

1 Comparison of the basic conditions

The patients with cervical erosion in the outpatient department arerandomly divided into two groups: 50 cases in trial group curing withthe preparation (embrocation) and 25 cases in the contrast group usingALBOTHYL. The basic conditions of the two groups are as the followingtable. TABLE 1 Comparison of the selective patients in age, menarcheage, pregnancy and delivery times Age Menarche Pregnancy times Deliverytimes Cases Mean SD Mean SD Mean SD Mean SD Trial group 50 35.88 6.8814.14 0.95 1.24 0.48 0.94 0.24 Contrast group 25 34.44 5.87 14.16 0.941.16 0.37 1.00 — Significance of the two P = 0.3737 P = 0.9315 P =0.4349 P = 0.2205 groups' variance

In the two groups, the youngest is 24 years old, and the oldest is 45years old. The basic conditions have no obvious significant variance.That is to say, they are comparable.

2. Clinical Manifestation

2.1 Conditions of the Two Patient Groups in Degree and Types of CervicalErosion TABLE 2 Comparison of the two patient groups in degree and typesof cervical erosion Pre-medication mod- The third re-examination milderate severe normal mild moderate severe Trial 21 24 5 44 6 — — groupContrast  8 10 7 19 5 1 — group

Significance test of the trial group before medication and at the thirdre-examination (exact probability) shows P<0.01, so does the contrastgroup (P<0.01).

As showed in table 2 the mild erosions take 42%, the moderate take 48%,the severe take 10% in the trial group. While in the contrast grouptheir percentages are separately 32%, 40% and 24%. After treatment, thenormal gets to 88%, the mild reaches 12%. Meanwhile in the contrastgroup, the normal takes 76%, the mild takes 20%, the moderate takes 4%,which obviously shows that the trial group is prior to the contrast one.TABLE 3 Comparison of the types of cervical erosion Pre-medication Thethird re-examination Simple granular papillary normal Simple granularpapillary Trial group 30 17 3 44 2 3 1 Contrast group 10 11 4 19 2 2 2

Note: In the tables, the normal refers to the filling of 0 in designingtreatment effects. Significance test of pre-medication and the thirdre-examination in the trial group (exact probability) shows P<0.01.

From table 3, it can be found that in the pre-medication trial groupsimple types takes 60%, granular types take 34% and papillary types take6%. In pre-medication contrast group, the three types show separately40%, 44% and 16%. In post-therapy trial group, they separately take 4%,6%, 2% and the normal takes 88%. In post-therapy contrast group, simpletypes take 4%, granular types take 4%, papillary types take 8% and thenormal takes 76%. TABLE 4 Change of leucorrhoea quantity andcharacteristics before and after medication first secondary thirdPre-medication re-exam re-exam re-exam Normal more much Normal more muchNormal more much Normal more much Trial group 27 21 2 45 4 1 50 — — 50 —— Contrast group 13 7 5 18 6 1 21 4 — 23 2 —

Significance test of the trial group after medication and at the thirdre-examination (exact probability) shows P<0.0 1. So does the contrastgroup(P<0.0 1).

From the above table, conclusions can be made that

(1) In the trial group, before therapy the patients with normalleucorrhoea quantity take 54%, the patients with a little moreleucorrhoea take 42%, the patients with much leucorrhoea take 4%.Meanwhile, in the contrast group, they take separately 52%, 28%, and20%.

(2) After therapy, the patients with normal leucorrhoea quantity take100%. In the contrast group the patients with normal leucorrhoeaquantity take 92%, with a little more leucorrhoea take 8%. There isobvious variance both in the trial and contrast group (P<0.01).

From the percentage of the patients in these two groups, the normaltakes 90% in the trial group, whereas in the contrast group they takeseparately 72% and 82%. TABLE 5 Comparison of leucorrhoeacharacteristics First Secondary Third Pre-medication re-examinationre-examination re-examination Normal Purulent Normal Purulent NormalPurulent Normal Purulent Trial group 44 6 48 2 49 1 49 1 Contrast group18 7 23 2 25 1 25—

Significance test of the trial group after medication and. at the thirdre-examination (exact probability) shows P<0.01. So does the contrastgroup (P<0.01).

Table 5 shows that (A) the patients with normal leucorrhoea in the trialgroup before therapy take 88%, with purulent leucorrhoea take 12%. Thepatients with normal leucorrhoea in the contrast group before therapytake 72%, with purulent leucorrhoea take 24%. After therapy, thepatients with normal leucorrhoea in the trial group take 98%, withpurulent leucorrhoea take 2%, and the patients with normal leucorrhoeain the contrast group take 100%. After therapy, the patients with normalleucorrhoea in the trial group take 98%, and in the contrast group theytake 100%. So conclusions can be made that the significance varianceexists between the two groups both before and after therapy, but doesnot exist between the trial and contrast group (P>0.05), which may bedue to the patients chief complaint of leucorrhoea quantity andcharacteristics.

2.3 Vaginal Clearance Degree, Cervical Smear and Papanicolaou Degree isShown in Table 6. TABLE 6 Vaginal clearance degree, cervical smear andpapanicolaou degree Vaginal clearance degree Papanicolaou degree Pre-third re- Pre- third re- medication examination medication examination III III I II III I II III I II III trial group 22 — 28 47 3 — 30 — 20 491 — contrast group 14 — 11 23 2 — 13 — 12 25 — —

By statistics, the P value in the two groups before and after therapyare both less than 0.01 in vaginal clearance degree and papanicolaoudegree by smearing. The obvious variance indicates the integrity andutility value of the preparation.

3. Therapy evaluation shown in table 7 TABLE 7 Therapy evaluation Healedeffective useless Trial group 44 6 0 Contrast group 19 6 0

In table 7, the healed rate in trial group is 88%, effective rate is12%, and common effective rate is 100%. Moreover in the contrast groupthey are separately 72%, 28% and 100%. By x² test the variance is notobvious.

4.Safety and Side Effects TABLE 8 side effects Common cases Yes No Trialgroup 50 0 50 Contrast group 25 0 25

There is even no side effect both in the fifty cases of trial group andthe twenty-five cases of contrast group, which indicates that thepreparation (embrocation) is safe and reliable in the clinic. TABLE 9The results of laboratory test before and after therapy Bb (g/L) RBC(10/L) WBC (10/L) BUN (mg/D1) Cr (mg/D1) Cases Mean ± SD Mean ± SD Mean± SD Mean ± SD Mean ± SD Trial group pre-medication 50 133.80 ± 9.023.95 ± 0.23 7.03 ± 1.95 4.45 ± 2.22 60.07 ± 18.49 post-medication 50137.56 ± 3.69 3.96 ± 0.46 7.29 ± 1.23 4.20 ± 1.55 65.85 ± 21.88Difference of P value P = 0.0014 P = 0.0806 P = 0.1104 P = 0.5527 P =0.1013 before and after therapy Contrast group pre-medication 25 137.76± 3.46 4.00 ± 0.15 6.80 ± 1.38 4.25 ± 1.26 74.82 ± 23.80 post-medication25 138.52 ± 4.45 4.03 ± 0.13 7.20 ± 1.24 3.88 ± 1.37 68.23 ± 24.48Difference of P value P = 0.1119 P = 0.4301 P = 0.3115 P = 0.3791 P =0.3205 before and after therapy

TABLE 10 Significance test of indexes in the trial and contrast groupbefore and after therapy Rb RBC WBC BUN Cr Before P = 0.0704 P = 0.3963P = 0.6048 P = 0.8684 P = 0.007 therapy After therapy P = 0.122  P =0.964 P = 0.7019 P = 0.5754 P = 0.7377Observation of the Indexes Before and After Medication

Many indexes such as RBC, WBC, aminotransferase, BUN, Cr are measuredbefore medication. Comparing the two groups with the indexes, most ofthem have no obvious variance (P>0.05). Though some indexes haveevaluation variance, the means are in the normal range (Detail in table9 and 10). This result suggests that the preparations admitted in thetrial and contrast groups are both safe.

6. X² significance of the indexes before and after medication TABLE 11X² significance of the indexes before and after medication degree oftypes of characters of Induced papanicolaou Massive cervical erosioncervical erosion leucorrhoea clearance degree degree Significance andvariance P < 0.05 P < 0.01 P < 0.01 P > 0.05 P < 0.01 P < 0.01 test inthe trial group before the third therapy (Exact probability)significance variance test P < 0.05 P < 0.01 P < 0.01 P > 0.01 P < 0.01P < 0.01 in the contrast group before therapy and the thirdre-examination (exact probability method) Significance variance test P >0.05 P > 0.02 P > 0.05 P > 0.05 P > 0.05 P > 0.05 of the trial andcontrast group before therapy (exact probability method) Significancevariance test P > 0.05 P > 0.02 P > 0.05 P > 0.05 P > 0.05 P > 0.05 ofthe trail and contrast group at the third re-examination (exactprobability method)

In a word, the two preparations both have obvious effects, especially inthe trial group for treatment of cervical erosion, which healed rate canreach 88% and total effective rate can reach 100%. They are both able toreduce the vaginal secretion and change its characteristics. At the sametime their side effects are also minor, causing no obvious localstimulation and no hurts to normal tissues. For the treatment ofcervical erosion, both of them are highly effective, safe and convenientexternal preparations. Meanwhile as to the reasonable administrationmanner, cotton ball with tail adheres erosive surface tightly and isconvenient to take down by the patient herself, the preparation isaccepted by the patients pleasantly.

In conclusion, the preparation (embrocation) provides a new way to cure

1. A preparation for treatment of cervical erosion, wherein comprisingproducts of Nocardia Rubra cell wall skeleton.
 2. The preparation ofclaim 1, wherein comprising pharmaceutically acceptable carriers.
 3. Thepreparation of claim 1, wherein Nocardia Rubra cell is deposited inChina General Microbiological Culture Collection Center with depositionNo.: CGMCC No0712.
 4. The preparation of claim 2, wherein thepharmaceutically acceptable carriers include carbohydrate, glycoside,fat or fatty acid, protein, amino acid, esters, spirits, demasorb,hydroxycarboxylic acid, poly-anion, surfactant, antiseptic, antioxidantand dextran.
 5. The preparation claim 2, wherein content of NocardiaRubra cell wall skeleton is in amount of 0.005 weight %-99 weight %based on the total weight of the preparation.
 6. A process and testmethod of a preparation of treating cervical erosion wherein: Aftercultivating, collecting thallus, cell fragmentation, purifying theenzyme, removing the grease, Nocardia Rubra cell wall skeleton isobtained, followed by mixing the Nocardia Rubra cell wall skeleton andthe pharmaceutically acceptable carriers to obtain the preparation; Aquality test method includes batch detection of strain of NocardiaRubra, culture liquid of Nocardia Rubra cell wall skeleton and a finalproduct containing Nocadia Rubra cell wall skeleton.
 7. The preparationof claim 6, wherein the pharmaceutically acceptable carriers includecarbohydrate, glycoside, fat or fatty acid, protein, amino acid, esters,spirits, demasorb, hydroxycarboxylic acid, poly-anion, surfactant,antiseptic, antioxidant and dextran.
 8. The preparation of claim 6,wherein the content of Nocardia Rubra cell wall skeleton is in amount of0.005 weight %˜99 weight % based on the total weight of the preparation.9. A method for treatment of cervical erosion, wherein applyingpharmaceutically effective amount of Nocardia Rubra cell wall skeletonpreparation directly on a suffering site of subject.